Human Gene Therapy Methods最新文献

Non-small cell lung cancer (NSCLC) denotes the most common type of lung cancers with high mortality globally. Long non-coding RNAs (lncRNAs) with differential expression have been indicated to be participants in the pathogenesis and development of cancer. However, the precise role of lncRNAs in NSCLC is still largely obscure. In this study, we explored a newly discovered intergenic lncRNA LINC00958 in NSCLC. First of all, the online databases suggested that LINC00958 was slightly expressed in human normal lung tissues but upregulated in LUSC tissues. Besides, the upregulation of LINC00958 in both lung adenocarcinoma (LUAD) and LUSC cell lines was easily found when compared with the normal BEAS-2B cells. In addition, we elucidated that knockdown of LINC00958 led to impaired proliferation, induced apoptosis, and hampered migration in LUAD cells. Moreover, a typical oncogenic pathway, JNK signaling, was verified to be involved in LINC00958-contributed LUAD development. Of note, we explained that LINC00958 exerted the tumor-promoting function in LUAD by enhancing the transactivation of p-c-JUN through activating JNK signaling. Meanwhile, we also revealed that LINC00958 was transcriptionally regulated by c-JUN. In addition, earlier findings were also suitable for LUSC cells. By and large, our work illustrated that LINC00958 facilitates tumorigenesis in NSCLC by activating the JNK/c-JUN signaling pathway, indicating a new road for diagnosis and treatment of both LUAD and LUSC.

Adeno-associated viral (AAV) vectors have shown great promise in gene delivery as evidenced by recent FDA approvals. Despite efforts to optimize manufacturing for good manufacturing practice (GMP) productions, few academic laboratories have the resources to assess vector composition. One critical component of vector quality is packaged genome fidelity. Errors in viral genome replication and packaging can result in the incorporation of faulty genomes with mutations, truncations, or rearrangements, compromising vector potency. Thus, sequence validation of packaged genome composition is an important quality control (QC), even in academic settings. We developed Fast-Seq, an end-to-end method for extraction, purification, sequencing, and data analysis of packaged single-stranded AAV (ssAAV) genomes intended for non-GMP preclinical environments. We validated Fast-Seq on ssAAV vectors with three different genome compositions (CAG-GFP, CAG-tdTomato, EF1α-FLuc), three different genome sizes (2.9, 3.6, 4.4 kb), packaged in four different capsid serotypes (AAV1, AAV2, AAV5, and AAV8), and produced using the two most common production methods (Baculovirus-Sf9 and human HEK293), from both common commercial vendors and academic core facilities supplying academic laboratories. We achieved an average genome coverage of >1,400 × and an average inverted terminal repeat coverage of >280 × , despite the many differences in composition of each ssAAV sample. When compared with other ssAAV next-generation sequencing (NGS) methods for GMP settings, Fast-Seq has several unique advantages: Tn5 transposase-based fragmentation rather than sonication, 125 × less input DNA, simpler adapter ligation, compatibility with commonly available inexpensive sequencing instruments, and free open-source data analysis code in a preassembled customizable Docker container designed for novices. Fast-Seq can be completed in 18 h, is more cost-effective than other NGS methods, and is more accurate than Sanger sequencing, which is generally only applied at 1-2 × sequencing depth. Fast-Seq is a rapid, simple, and inexpensive methodology to validate packaged ssAAV genomes in academic settings.

Viral vectors are complex drugs that pose a particular challenge for manufacturing. Previous studies have shown that, unlike small-molecule drugs, vector preparations do not yield a collection of identical particles. Instead, a mixture of particles that vary in capsid stoichiometry and impurities is created, which may differ from lot to lot. The consequences of this are unclear, but conflicting reports regarding the biological properties of vectors, including transduction patterns, suggest that this variability may have an effect. However, other variables, including differences in animal strains and techniques, make it difficult to identify a cause. Here, we report lot-to-lot variation in spinal cord gray matter transduction following intrathecal delivery of self-complementary adeno-associated virus serotype 9 vectors. Eleven lots of vector were evaluated from six vector cores, including one preclinical/Good Laboratory Practice lot. Eight of the lots, including the preclinical lot, failed to transduce the gray matter, whereas the other three provided robust transduction. The cause for this variation is unknown, but it did not correlate with vector titer, buffer, or purification method. These results highlight the need to identify the cause of this variation and to develop improved production and quality control methods to ensure lot-to-lot consistency of vector potency.